Separation and purification of biocomponents and drugs are commonly performed by ion-exchange chromatography, reverse phase chromatography, affinity chromatography and the like. Of them, the affinity chromatography is the only method for effective purification of almost all biomolecules based on their biological functions or individual chemical structures and is a very unique separation technique.
Affinity chromatography is one type of adsorption chromatography in which the substance to be purified is specifically and reversibly adsorbed onto a bound substance (ligand) which has been immobilized on an insoluble carrier (matrix). It is widely applied to the separation and purification of biological substances since most separations can be accomplished in one stage and a remarkable time saving can be achieved compared to a multi-stage method with low in selectivity.
However, according to the common affinity chromatography, the salt concentration, the organic solvent concentration, the pH and the like of an eluent are varied in the elution of the biocomponents, drugs, recombinant proteins and the like which come to target substances or a gradient of a substance to be competitively bonded to the ligand is used. It is known that such methods are sometimes reduced in recovery since the conditions such as the salt concentration and the organic solvent concentration often become severe with respect to the target substance. Furthermore, the salt, the organic solvent and the substance to be competitively bonded to the ligand which have been used in the elution of the target substance must finally be removed by operations such as desalting and drying but, when the substance to be competitively bonded to the ligand and the target substance are of a similar molecular size, it is very difficult or sometimes impossible to separate the target substance from the substance used in the elution. In addition, the activity and the recovery of the final substance are often significantly reduced by the operations of desalting and drying.
In eluting a target substance, the elution by changing the composition of an eluent such as the salt, the organic solvent, the pH, the concentration of the substance to be competitively bonded to a ligand can cause the above described problems such inactivation, reduction in recovery and the like by the use of chemical substances including, for example, salts, organic solvents, acids and bases in the eluent and substances to be competitively bonded to ligands. However, if it is possible to create an environment which affects the elution of a target substance not by such chemical methods but by physical changes such as heat, light and magnetism, it would be possible to elute the target substance by a physical means to solve the problems of inactivation and reduction in recovery and the like.
Recently, the description relating to separation materials comprising stimulus responsive polymers covalently bonded to ion-exchange groups has been found (see, for example, Japanese Patent Application No. Hei 10-140722 and its counterpart WO 99-61904).
Galaev et al., Journal of Chromatography A684 (1994) 37-43 describe the elution by temperature of lactate dehydrogenase (LDH) in a chromatographic system using a base matrix to which Cibacron Blue is covalently bonded and on which a temperature responsive polymer (polyvinyl caprolactam) is also physically adsorbed. However, the clouding point of the polyvinyl lactam is 38° C. and it is necessary to raise temperature up to 40° C. in adsorbing the polyvinyl lactam on the base matrix and thus, there is a fear of causing denaturation depending on the target protein used. In general, the clouding point can be lowered by introducing a hydrophobic group but in this instance, it can be anticipated that the recovery will be decreased by an increase of non-specific adsorption on the hydrophobic group.
Hofman et al., (WO 87/06152) describe a separation method using a ligand bonded to a temperature responsive polymer. Furthermore, in WO 97/09068 there is proposed a separation method by molecular conjugates obtained by introducing a stimulus responsive polymer into a substance having specific affinity for a target substance.
Further, in Japanese Patent Publication (Kokai) No. Hei 7-135957/1995 and WO 97/09068 there are described separation materials constituted by a substance having specific affinity for a target substance and a stimulus responsive polymer.
However, none of these patents suggest a technique for increasing the efficiency of the elution by temperature by immobilizing a substance having affinity for a target substance and a low molecular substance which competitively binds to a ligand.
There are also a number of publications which describe chromatography based on separation materials comprising stimulus responsive polymers which do not have a ligand covalently bonded to temperature responsive polymers. Gewehr et al. [Macromolecular Chemistry and Physics 193 (1992) 249256] describe gel chromatography on porous silica beads coated with a stimulus responsive polymer. Hosoya et al. [Anal. Chem. 67 (1995) 1907-1911]; Yamamoto et al. [Proc. 114th National Meeting of the Pharmaceutical Society of Japan, Tokyo (1994) 160]; Kanazawa et al. [Yakugaku Zasshi 117 (10-11) (1997) 817-824]; Kanazawa et al. [Anal. Chem. 68(1) (1996) 100-105]; Kanazawa et al. [Anal. Chem. 69(5) (1997) 823-830]; Kanazawa et al. [J. Pharm. Biomed. Anal. 15 (1997) 1545-1550]; Yakushiji et al. [Langmuir 14(16) 1998) 4657-466268]; Kanazawa et al. [Trends Anal. Biochem.17 (7) (1998) 435-440]; Yakushiji et al. [Anal. Chem. 71(6) 1999]1125-1130); Grace & Co. (EP 534016); and Okano [Japanese Patent Publication (Kokai) No. Hei 6-108643/1994] all describe reversed phase chromatography on a matrix coated with a heat responsive polymer for aiming to separate biopolymers. The matrix can be porous. The utilized hydrophobic groups are possessed by the polymer as such. In these prior art documents there is no description of ligands covalently bonded to the polymer after polymerization
Furthermore, the following various separation materials using stimulus responsive polymers are proposed.
In Japanese Patent Publication No. Hei 7-136505/1995 there is a description that with the use of an affinity separation material in which a substance having specific affinity for a target substance is bonded to a stimulus responsive polymer through a spacer comprising a bound substance composed of two compounds having bindability to each other (for example, avidin-biotin), the dissociation between the two compounds is caused by a stimulus (for example, temperature) to recover the target substance. However, acceleration of sufficient dissociation cannot be obtained by the structural change of the polymer due to the stimulus and in the case of cells the recover is as low as about 50%.
In Japanese Patent Publication (Kokai) No. Hei 8-103653/1996 and Japanese Patent Publication (Kokai) No. Hei 9-49830/1997 there is a description relating to a separation/purification method characterized in that in the stimulus responsive separation material having a region composed of a stimulus responsive polymer chain and a region having affinity for a target substance in the surface of a base matrix, after the target substance is adsorbed on the separation material, the target substance is released from the separation material by varying the structure of higher order of the stimulus responsive polymer. However, also in this instance, acceleration of sufficient dissociation by the structural change of the polymer due to a stimulus cannot be obtained and thus, the recovery is as low as 63% even in the case of cells.
In WO 94-15951 is described a method of separating a substance with the use of an affinity separation material using a composite ligand comprising a ligand bonded to a target substance having bindability weaker than the bindability of the first target substance. Also, this publication describes use of a stimulus responsive polymer but does not suggest simultaneous use of a lower-molecular substance.
However, according to the methods described in these publications, it is difficult to purify a target substance with a high recovery without changing the composition of an eluent. When the target substance to be purified, particularly by a lectin affinity chromatography, is a low-molecular compound such as a sugar chain, use of the lectin affinity chromatography is sometimes limited due to the difficulty in separating the sugar chain from a hapten sugar if the hapten sugar is used in the elution. Furthermore, there have been problems in increasing detection sensitivity and regenerating carriers.